Regulation and biological effect of endogenous insulin-like growth factor binding protein-5 in human osteoblastic cells

J Clin Endocrinol Metab. 1993 May;76(5):1153-9. doi: 10.1210/jcem.76.5.7684391.

Abstract

U-2 human osteosarcoma cells secrete a 29/32/34 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) identified as O-glycosylated IGFBP-5. Treatment of U-2 cells with IGF-I markedly increased medium levels of IGFBP-5 in a concentration- and time-dependent manner; other skeletal regulatory factors (GH, insulin, PTH, dexamethasone, beta-estradiol, 1,25-dihydroxyvitamin D3, transforming growth factor-beta) had no effect. IGF-I increased IGFBP-5 levels in the culture medium 10-fold without influencing IGFBP-5 messenger RNA abundance. IGF-I, IGF-II, and the IGF-I analog [1-27Gly(4)38-70] IGF-I bound IGFBP-5 with high affinity and, when added to U-2 cultures, effectively promoted IGFBP-5 accumulation in the medium. On the other hand, des(1-3)IGF-I and [QAYL]IGF-I, IGF-I analogs that did not bind IGFBP-5, failed to elicit an increase in medium IGFBP-5. Cell-free incubation of recombinant human (rh) IGFBP-5 in U-2 conditioned medium resulted in a marked reduction of detectable rhIGFBP-5; the presence of IGF-I or IGF-II peptide partially prevented this decrease. By immunoblot analysis, loss of intact rhIGFBP-5 (29-kDa unreduced, 34-kDa reduced) coincided with the appearance of a 16-kDa proteolytic fragment. U-2 conditioned medium contained immunoreactive IGFBP-5 at 29-34-kDa, 20-kDa, 17-kDa, and 16-kDa. Endogenous IGFBP-5 inhibited IGF-I but not des(1-3)IGF-I-stimulated U-2 cell proliferation. In conclusion, IGF peptides can regulate the availability of IGFBP-5 in osteoblast-like cells by impeding IGFBP-5 proteolysis. The biological consequence of increased medium IGFBP-5 appears to be decreased cell responsiveness to IGF-I stimulation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blotting, Northern
  • Blotting, Western
  • Carrier Proteins / metabolism*
  • Dose-Response Relationship, Drug
  • Humans
  • Insulin-Like Growth Factor Binding Protein 5
  • Insulin-Like Growth Factor I / metabolism
  • Insulin-Like Growth Factor I / pharmacology
  • Insulin-Like Growth Factor II / metabolism
  • Insulin-Like Growth Factor II / pharmacology
  • Osteoblasts / metabolism*
  • Precipitin Tests
  • Somatomedins / metabolism
  • Tumor Cells, Cultured

Substances

  • Carrier Proteins
  • Insulin-Like Growth Factor Binding Protein 5
  • Somatomedins
  • Insulin-Like Growth Factor I
  • Insulin-Like Growth Factor II